The broad, long-term objectives of the project are to characterize the structure, regulation and function of the protein tyrosine phosphatase (PTP) family of enzymes. It is now apparent that the coordinated and competing actions of both protein tyrosine kinases (PTKs) and PTPs are integrated in vivo to control such fundamental processes as growth and proliferation, differentiation, survival, motility and metabolism. Furthermore, disruption of the delicate balance between the action of PTPs and PTKs has been implicated in the etiology of human diseases, including cancer, diabetes and inflammation. Therefore, characterization of the PTPs is a prerequisite to gaining a complete understanding of the physiological consequences of tyrosine phosphorylation under normal and diseased conditions. This competitive renewal focuses on a new tier of control of tyrosine-phosphorylation dependent signal transduction, the regulation of PTP function by reversible oxidation. The Specific Aims of the proposal are: 1) To develop new strategies to assay reversible oxidation of PTPs and to determine the stoichiometry of oxidation in response to physiological stimuli. 2) To investigate the interplay between PTP oxidation and the control of pTyr-dependent signaling. 3) To investigate mechanisms underlying the specificity of stimulus-induced PTP oxidation. 4) To investigate the potential function of the second PTP domain in Receptor PTPs as an oxidation sensor. New assays will be developed to measure the reversible oxidation of PTPs in response to physiological stimuli. Furthermore, such oxidation will be harnessed as a means of "tagging" the specific PTPs that are integral to the regulation of signal transduction pathways initiated by those stimuli. By combining the use of RNA interference with application of substrate trapping mutant forms of the PTPs, the signaling function of these enzymes will be defined. It is anticipated that the insights that are generated may identify novel targets for therapeutic intervention in human disease and may suggest new therapeutic strategies for PTP inhibition.